Ki67 IHC vs Real-time PCR - studying proliferation (May/01/2008 )

I want to look at proliferation of some cells.

I will do tritiated thymidine uptake first, but then I am thinking about doing Ki67 as a second experiment to confirm the finding.

The question is:

Is it necessary to using anitbody to stain the cells and look at the presence of protein?
I've done immunostaining before and I have experience, the problem is now I am in a new lab that doesn't do a lot of these things and I'll have to buy stuff, like Ki67 Antibody, 2nd FITC Antibody, DAPI, etc.

Could I just extract RNA and do real-time PCR to look at Ki67 mRNA level??
I don't see why I couldn't, but most literature I found used immunostain that makes me a bit nervous.



Any comments and personal experience will be much appreciated, thanks!!



Julian Ph.D.
NICHD Fellow

can you get single cells? how about a simple PI staining and then do FACS? that will give you a basic cell cycle profile, and is quick and easy and not requiring much reagents other than some PI dye. If that works fine and when you guys can buy more stuff, you can combine that with some BrdU labeling to better quantify your S phase population.

Good luck! I miss doing cell biology a lot.